High‐throughput lipidomic profiles sampled with electroporation‐based biopsy differentiate healthy skin, cutaneous squamous cell carcinoma, and basal cell carcinoma

Abstract Background The incidence rates of cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) skin cancers are rising, while the current diagnostic process is time‐consuming. We describe the development of a novel approach to high‐throughput sampling of tissue lipids using electroporation‐based biopsy, termed e‐biopsy. We report on the ability of the e‐biopsy technique to harvest large amounts of lipids from human skin samples. Materials and Methods Here, 168 lipids were reliably identified from 12 patients providing a total of 13 samples. The extracted lipids were profiled with ultra‐performance liquid chromatography and tandem mass spectrometry (UPLC‐MS‐MS) providing cSCC, BCC, and healthy skin lipidomic profiles. Results Comparative analysis identified 27 differentially expressed lipids (p < 0.05). The general profile trend is low diglycerides in both cSCC and BCC, high phospholipids in BCC, and high lyso‐phospholipids in cSCC compared to healthy skin tissue samples. Conclusion The results contribute to the growing body of knowledge that can potentially lead to novel insights into these skin cancers and demonstrate the potential of the e‐biopsy technique for the analysis of lipidomic profiles of human skin tissues.

will prove useful in light of incidence trends.Methods that can provide rapid results will also be beneficial considering the difference in aggressiveness and likelihood of metastasis of cSCC and BCC. 9 Collecting samples of molecular information from cSCC and BCC lesioned skin with modern technology like e-biopsy and others 10,11 is simpler and faster than current biopsy methods, alluding to the potential of molecular profiling in diagnostics.cSCC and BCC are cancers of keratinocytes that functionally make and secrete lipids, 12 , therefore, exploring the differential expression of their lipid profiles may reveal trends that differentiate the two from each other as well as from healthy skin.
Molecular profiling technology gives us snapshots of the cellular fabric where we can observe trends that have physical manifestations.[18][19][20] Lipids profiles, however, have mostly been reported for BCC [21][22][23] using low-throughput methods and serum samples with the exception of one high-throughput cSCC study that included 70 lipids among metabolites. 24Beyond medicine, molecular profiling plays an important role in other aspects of life such use in agriculture food and safety, forensic science, and environmental monitoring.
[27] Coupling this sampling method with UPLC-MS-MS, which has proven its ability to identify potential markers of skin cancer, 18 provides a promising direction for high-throughput analysis of extractable molecules.
Here we perform a comparative analysis of the first high-throughput lipidomic profiling of cSCC, BCC, and healthy skin tissue using e-biopsy.[27]

Human patients
A list of patient age, sex, and tumor type is provided in Table 1.
This study was approved by the Meir Medical Center IRB, number MMC-19-0230.All patients gave consent for participation and for the performance of genetic analysis of their sample tissue.Figure 1 summarizes the workflow and e-biopsy method.

Sample collection
E-biopsy was performed with a custom-made high-voltage pulsed electric field generator under conditions previously described for protein extraction from cSCC and BCC. 16The liquids sampled were immediately transferred to 1.5 mL tubes with 100 µL double distilled water and stored at −20 • C until shipped to Beijing Genomics Institute for analysis.

Differential lipid screening
The measured lipid intensities (

Volcano plot analysis
Volcano plot overlays the magnitude of the fold change of lipids with their differential significance between two analyzed populations.Differentially expressed lipids were defined for this analysis as those with a -log10(p-value) > 1.3 (i.e., p-value < 0.05) and those with −1 < log2(foldchange) < 1. Fold change calculations were carried out using the average of intensity values for each comparison group, that is, foldchange(lipid) = avg(grp1) / avg(grp2).The data was then filtered to include only the compounds with high-reliability scores (grades A and B) (Figure 3).The data of the most interesting compounds were compiled into tables to showcase their associated p-values and fold change values (Table 2).

RESULTS
The initial 309 identified lipids were filtered according to reliability  2, Tables S2-S4.
over-and under-expressed lipids.The major findings are summarized in Table 2.

Electroporation sampled lipidomic profile differentiate BCC vs. Healthy skin
The overabundance analysis of BCC compared to Healthy showed a total of 17 lipids with Student's T-test p-values less than 0.05 (Figure 2B, corresponding to FDR = 0.49).Moreover, six of these lipids resulted in p-value below 1e-2 (FDR = 0.28) and four of them in p-value below 1e-3 (FDR = 0.04).Of these, eight were significantly lower in BCC and seven significantly higher in BCC (Figure 3B).

DISCUSSION
We performed a comparison of high-throughput lipidomic profiles sampled with e-biopsy from healthy, cSCC, and BCC skin tissues.
The overabundance and volcano plot analyses suggest a difference in lipidomic profiles between cancer and healthy skins, with a general trend of lower DGs and higher phospholipid subclasses in cancerous tissue.There was also a slight difference and a separation potential between cSCC and BCC lipid profiles as higher intensities of phospholipids and other lipids were observed in cSCC.The comparison of cSCC to healthy tissue revealed lower DGs and higher phospholipids and lyso-phospholipids.Similarly, the comparison of BCC to healthy skin found lower diglycerides and higher phospholipids.In the BCC to healthy tissue comparison, two ceramides were identified, one higher and the other lower in BCC.In the comparison of cSCC to BCC, several lyso-phospholipids, and a single phospholipid, ceramide, and triglyceride were identified at higher intensities in cSCC.
To the best of our knowledge, no previous molecular profiling study focused only on the lipidomic profiling of cSCC.Previous studies comparing BCC and healthy (with sample sizes of 30 and 64) 21,23 and a study comparing 12 BCC, 13 AK, and 11 healthy skin samples, 22 reported lipidomic profiles for only six lipid groups: cholesterol, HDL, LDL, triglycerides, phospholipids, and total lipids.2][23] Phospholipids were previously found significantly higher in BCC versus Healthy in both skin and serum samples. 22r study significantly improves on the level of detail of reported lipids of BCC and healthy skin.E-biopsy coupled with UPLC-MS-MS was able to measure specific ceramides, lyso-phospholipids, and diglycerides in addition to triglycerides and phospholipids.In contrast to a previous report, 22 triglycerides were not identified as significant in BCC compared to Healthy skin samples.Rather, they were found in significantly lower levels in BCC compared to cSCC.Like in the previous study, 22 our results show higher phospholipid levels in BCC compared to Healthy skin.Six phospholipid subclasses were expressed higher in BCC compared to Healthy, and one phospholipid type was lower in BCC compared to Healthy (Figure 3B).
Our study contributes novel information on the lipid profile of cSCC and its comparative analysis to BCC and to healthy skin.Diglycerides, triglycerides, ceramides, phospholipids, and lyso-phospholipids were identified in cSCC.Diglycerides were expressed significantly lower and phospholipids and lyso-phospholipids were significantly higher in cSCC compared to healthy skin (Figure 3A).In cSCC compared to BCC, phospholipids, lyso-phospholipids, ceramides, and triglycerides were expressed significantly higher (Figure 3C).This study differs from previous cSCC studies in the methods of sample collection and lipid analysis.Previously, cSCC and healthy skin were sampled from the same patient with healthy skin taken from beyond tumor margins, and lipids were analyzed among metabolites from whole tissue samples. 24is study, in contrast, collected healthy skin tissue from a separate set of patients and performed an exclusive lipid analysis on the collected tissues.This resulted in a larger number of lipids detected and analyzed and better reflects the real-life diversity of the human lipidome.Additionally, the whole tissue samples in previous studies were collected over relatively large areas, thus allowing the inherent tissue and tumor molecular heterogeneity to obscure the signals of interest.This is in contrast to localized sampling using e-biopsy which better reflects the actual spatial molecular state in the condition of interest.
The lipids included in the molecular profiling, have variable reported effects on carcinogenesis.Ceramides (Cer) are potent tumor suppressor lipids as they can enhance keratinocytes apoptosis and also block cell cycle transition limiting cancer cell proliferation. 29Diglycosylceramides (CerG2) are involved in forming large amounts of lipids in the cells of the innate immune system. 29The higher Cer levels in cSCC compared to BCC may be a reflection of the difference in aggressiveness and the need for stronger tumor suppression in cSCC.Higher CerG2 levels in BCC compared to healthy suggest an immune response is occurring in the tumor area.Phosphatidylserine (PS) is translocated from the inner to outer endothelial cell's membrane when exposed to oxidative stress, making it a potential marker for apoptotic and tumor cells. 30Phosphatidylcholine (PC) is involved in tumor microenvironment cellular communication and interestingly it was demonstrated that cancer cells accumulate PC precursors or products, compared to non-malignant counterparts. 31Similarly, increased triglycerides (TG) levels were seen in both actinic keratosis and BCC compared to normal skin cells. 223][34][35] Lyso-phosphatidylcholine (LPC) is mainly derived from the turnover of phosphatidylcholine (PC) in circulation by phospholipase A2 (PLA2). 36LPC can also induce the activation of PKC as well as phospholipase and regulate MAP kinase. 36LPC can recruit phagocytes to the site of apoptosis, hence plays an important role in the invasion, metastasis, and prognosis of tumors. 36This aligns with our findings of higher PC and LPC in cancer groups compared to healthy skin samples.Additionally, LPC was higher in cSCC compared to BCC, potentially reflecting the more aggressive nature of cSCC.Lyso-phosphatidylinositol (LPI) is generated by PLA2 and a G protein receptor 55 (GPR55), upregulated in human cSCC and suggested to promote skin carcinogenesis and tumor aggressiveness. 37[40] This supports the observed increase if PI in all comparison groups of this study (cSCC vs. healthy, BCC vs. healthy, and cSCC vs. BCC).
Last, Lyso-phosphatidylethanolamine (LPE)'s physiological significance in the plasma remains unknown, 30 however, it has been observed to significantly increase cell proliferation in breast cancer cell lines. 41ere are a few limitations in this study to be noted.First, the sample size was not representative enough to draw a confident conclusion on the specific lipid behavior.Previous studies demonstrate the need for larger sample numbers to observe trends in skin cancer lipids, 21,23 thus an increased sample size can improve differential expression analysis, increase the confidence in findings, and reduce the amount of falsely detected signals.Second, the study was performed on the exvivo samples, in-vivo sampling of skin may provide slightly different results.Third, the inclusion of patients in the study was not very strict, and patient lifestyle information that could provide useful information, such as levels of sun exposure, was not collected.
The e-biopsy sampling technique is still in its early stages but has the potential to be implemented in a handheld device, offering a promising solution to reduce the need for resection during biopsy.Unlike existing handheld devices like the iKnife 42,43 and MasSpec Pen, 44,45 which require a real-time connection to a mass spectrometer and are primarily used intraoperatively, the e-biopsy technique shows promise for broader applications.Other needle biopsy methods such as fine-needle aspiration and core needle biopsy also eliminate the need for resection but are limited by needle size, 46,47 and the need for increasing diagnostic accuracy requires more invasive procedures with larger needle diameters. 48,49In contrast, the e-biopsy technique has demonstrated the ability to sample areas larger than an actual needle diameter, 27 providing valuable site-specific information.This also stands in contrast to previous lipid analysis studies that relied on serum samples, lacking a specific spatial context.This site-specific sampling feature is advantageous as it enables the mapping of tumor heterogeneity and offers a deeper understanding of tumor complexities. 26

CONCLUSION
This study significantly contributes to the understanding of cSCC and BCC diagnostics.It provides the first of its kind report of cSCC high throughput lipidomic profiles.It also provides high throughput lipidomic profiles for BCC and healthy skin samples, together with comparative analysis between all three tissues.Moreover, all lipidomic profiles reported here are of greater resolution compared to previous keratinocyte carcinoma lipid profiling.A total of 168 lipids were identified, of which 27 were recognized as differentially expressed

F I G U R E 1
(A, B) E-biopsy analysis and workflow.(A) Sample collection through pulsed electric field (PEF) application leads to the extraction of water-soluble compounds that underwent subsequent UPLC-MS-MS and differential expression analysis.(B) Needle and electrode positioning on the skin during molecular harvesting by e-biopsy.F I G U R E 2 (A-C) Overabundance plots comparing the distribution of lipid differential expression (both over-and under-expression) p-values between control (normal skin tissue), BCC, and cSCC tumor samples.A total of 13 samples and 168 lipids extracted by e-biopsy were analyzed.The vertical red line marks the Student's T-test p-value cut-off of 0.05.(A) cSCC versus Healthy, (B) BCC versus Healthy, and (C) cSCC versus BCC.

2. 4 . 1
Statistical overabundance analysisOverabundance analysis compares actual and expected distributions of p-values to verify that compounds have different abundance levels when compared to classes of samples.28This approach explores internal data variability and helps address multiple comparisons.The analysis relies only on the number of compounds (i.e., lipids) and their observed corresponding p-values (here obtained from the Student's Ttest).The distribution of the expected p-values was generated from a null model assuming the same number of compounds (Figure2).
score, omitting lipids with grades C and D, which resulted in 168 lipids eligible for differential expression analysis.The analysis was performed for each of three comparison configurations: cSCC versus Healthy, BCC versus Healthy, and cSCC versus BCC.Overabundance plots (Figure 2) represent analysis results, where the number of lipids with Student's Ttest p-value below 0.05 is highlighted in red.Volcano plots (Figure 3) show relative group affinity of each lipid, highlighting significantly F I G U R E 3 (A-C) Volcano plots and tables showing the fold change difference of lipid intensities.(A) cSCC vs. Healthy.(B) BCC versus Healthy.(C) cSCC versus BCC.Tables with lipid names correspond to lettered data points in the adjacent volcano plots.Fold change and p-value data for lipids listed in the tables can be found in Table positive fold change): LPC(20:1), PC(16:0/16:1), LPE(18:0), LPC(16:0), PI(18:0/18:2), LPC(16:0)(rep), LPI(18:0), PC(16:0/14:0), LPC(14:0), and LPC(18:0e) (Figure3A).The high-resolution volcano plot of this comparison can be viewed in Figure S1.All associated p-values and fold change values for the lipids listed are reported in Table

Figure S3 .
Figure S3.All associated p-values and fold change values for the lipids listed are reported in TableS4.
in at least one comparison group.The differently expressed lipids are a variety of diglycerides, triglycerides, ceramides, phospholipids, and lyso-phospholipids.Overall trends indicate lower diglycerides and higher phospholipids and lyso-phospholipids in cSCC and BCC compared to Healthy skin tissue.In summary, this study significantly advances our understanding of cSCC and BCC diagnostics through high-throughput lipidomic profiling.The identification of differentially expressed lipids and the comparative analysis among the three tissue types provide crucial insights into the lipidomic alterations associated with these skin cancers.The availability of the data online and the potential of the e-biopsy technique pave the way for improved diagnostic approaches and hold promise for the future of skin cancer diagnosis.
Patient sex, age, and tumor type.
From March 2020 to March 2022, 10 tissue samples were collected from 10 patients who underwent surgical excision of a skin lesion suspected as BCC or cSCC at Meir Medical Center, Israel.Three healthy tissue samples were collected from two patients undergoing blepharoplasty.Excised samples were at least 1 cm in diameter.TA B L E 1E-biopsy extraction was performed on 13 fresh (between 10-20 min after surgery) samples.Lipid analysis was performed via UPLC-MS-MS.

TA B L E 2
Differentially expressed lipids with associated p-values and fold change values.Columns contain the lipids (in bold) identified as significant by the pairwise comparison of groups: CSCC vs. Healthy, BCC vs. Healthy, cSCC vs. BCC.Also categorized are the lipids found in combined cSCC and BCC groups compared to Healthy, showing the difference between keratinocyte carcinoma and healthy skin.Last, lipids of cSCC are isolated with results of common lipids that differentiate cSCC from BCC and healthy skin.Reading example: Cer(d18:2/24:1) has Student's T-test p-value of 0.01 and the ratio of its average intensity in BCC tissues to its average intensity in healthy tissues is 0.44.